Federal Register / Vol. 46, No. 233 / Friday, December 4, 1981 / Notices 
59405 
Supplement to the NIH Guidelines for 
Recombinant DNA Research." 
II-D. Biological Containment. 
II-D-1. Levels of Biological 
Containment. In consideration of 
biological containment, the vector 
(plasmid, organelle, or virus) for the 
recombinant DNA and the host 
(bacterial, plant, or animal cell) in which 
the vector is propagated in the 
laboratory will be considered together. 
Any combination of vector and host 
which is to provide biological 
containment must be chosen or 
constructed so that the following types 
of "escape” are minimized: (i) Survival 
of the vector in its host outside the 
laboratory and (ii) transmission of the 
vector from the propagation host to 
other nonlaboratory hosts. 
The following levels of biological 
containment (HV, or //ost- Sector, 
systems) for prokaryotes will be 
established: specific criteria will depend 
on the organisms to be used. Eukaryotic 
host-vector systems are considered in 
Part III. 
n-D-l-a. HVl. A host-vector system 
which provides a moderate level of 
containment. Specific systems: 
II-D-l-a-(l). EKl. The host is always 
E. coli K-12 or a derivative thereof, and 
the vectors include nonconjugative 
plasmids (e.g., pSClOl, ColEl, or 
derivatives thereof[21-27]) and variants 
of bacteriophage, such as lambda[28- 
33]. The E. coli K-12 hosts shall not 
contain conjugation-proficient plasmids, 
whether autonomous or integrated, or 
generalized transducing phages, except 
as specified in Section III-O. 
II-D-l-a-(2). Other Prokaryotes. 
Hosts and vectors shall be, at a 
minimum, comparable in containment to 
E. coli K-12 with a nonconjugative 
plasmid or bacteriophage vector. The 
data to be considered and a mechanism 
for approval of such HVl systems are 
described below (Section II-D-2). 
II-D-l-b. HV2. These are host-vector 
systems shown to provide a high level of 
biological containment as demonstrated 
by data from suitable tests performed in 
the laboratory. Escape of the 
recombinant DNA either via survival of 
the organisms or via transmission of 
recombinant DNA to other organisms 
should be less than Vio* under specified 
conditions. Specific systems: 
II-D-l-b-(l). For EK2 host-vector 
systems in which the vector is a 
plasmid, no more than one in 10“ host 
cells should be able to perpetuate a 
cloned DNA fragment under the 
specified nonpermissive laboratory 
conditions designed to represent the 
natural environment, either by survival 
of the original host or as a consequence 
of transmission of the cloned DNA 
fragment. 
II-D-l-b-(2). For EK2 host-vector 
systems in which the vector is a phage, 
no more than one in 10* phage particles 
should be able to perpetuate a cloned 
DNA fragment under the specified 
nonpermissible laboratory conditions 
designed to represent the natural 
environment either (i) as a prophage (in 
the inserted or plasmid form) in the 
laboratory host used for phage 
propagation or (ii) by surviving in 
natural environments and transferring a 
cloned DNA fragment to other hosts (or 
their resident prophages). 
II-D-l-c. HV3. These are host-vector 
systems in which: 
II-D-l-c-(l). All HV2 criteria are met. 
II-D-l-c-{2). The vector is dependent 
on its propagation host or is highly 
defective in mobilizability. Reversion to 
host-independence must be less than 
Vio* per vector genome per generation. 
n-D-l-c-(3). No markers conferring 
resistance to antibiotics commonly used 
clinically or in agriculture are carried by 
the vector, imless expression of such 
markers is dependent on the 
propagating host or on unique 
laboratory-controlled conditions or is 
blocked by the inserted DNA. 
II-D-l-c-(4). The specified 
containment shown by laboratory tests 
has been independently confirmed by 
specified tests in animals, including 
primates, and in other relevant 
environments. 
II-D-l-c-(5). The relevant genotypic 
and phenotjqjic traits have been 
independently confirmed. 
II-D-2. Certification of Host-Vector 
Systems. 
n-D-2-a. Responsibility. HVl 
systems other than E. coli K-12, and 
HV2 and HV3 host-vector systems, may 
not be designated as such imtil they 
have been certified by the Director, NIH. 
Application for certification of a host- 
vector system is made by written 
application to the Office of Recombinant 
DNA Activities, National Institutes of 
Health, Bethesda, Maryland 20205. 
Host-vector systems that are proposed 
for certification will be reviewed by the 
National Institutes of Health (NIH) 
Recombinant DNA Advisory Committee 
(RAC). (See Section IV-E-l-b-(l).) This 
will first involve review of the data on 
construction, properties, and testing of 
the proposed host-vector system by a 
Working Group composed of one or 
more members of the RAC and other 
persons chosen because of their 
expertise in evaluating such data. The 
Committee will then evaluate the report 
of the Working Group and any other 
available informaton at a regular 
meeting. The Director, NIH, is 
responsible for certification after 
receiving the advice of the RAC. Minor 
modifications of existing certified host- 
vector systems, where the modifications 
are of minimal or no consequence to the 
properties relevant to containment may 
be certified by the Director, NIH, 
without review by the RAC. (See 
Section IV-E-l-b-(3)-(f).) 
When new host-vector systems are 
certified, notice of the certification will 
be sent by the Office of Recombinant 
DNA Activities (ORDA) to the applicant 
and to all Institutional Biosafety 
Committees (IBCs) and will be 
published in the Recombinant DNA 
Technical Bulletin. Copies of a list of all 
currently certified host-vector systems 
may be obtained from ORDA at any 
time. 
The Director, NIH, may at any time 
rescind the certification of any host- 
vector system. (See Section FV-E-l-b- 
(3)-(i)0 If certification of a host-vector 
system is rescinded, NIH will instruct 
investigators to transfer cloned DNA 
into a different system, or use the clones 
at a higher physical containment level 
unless NIH determines that the already 
constructed clones incorporate adequate 
biological containment. 
Certification of a given system does 
not extend to modifications of either the 
host or vector component of that system. 
Such modified systems must be 
independently certified by the Director, 
NIH. If modifications are minor, it may 
only be necessary for the investigator to 
submit data showing that the 
modifications have either improved or 
not impaired the major phenotypic traits 
on which the containment of the system 
depends. Substantial modifications of a 
certified system require the submission 
of complete testing data. 
II-D-2-b. Data To be Submitted for 
Certification. 
II-D-2-b-(l). HVl Systems Other than 
E. Coli K-12. TTie following types of data 
shall be submitted, modified as 
appropriate for the particular system 
under consideration, (i) A description of 
the organism and vector; the strain’s 
natural habitat and-growth 
requirements; its physiological 
properties, particularly those related to 
its reproduction and survival and the 
mechanisms by which it exchanges 
genetic information; the range of 
organisms with which this organism 
normally exchanges genetic information 
and what sort of information is 
exchanged; and any relevant 
information on its pathogenicity or 
toxicity, (ii) A description of the history 
of the particular strains and vectors to 
be used, including data on any 
mutations which render this organism 
[294] 
